DETERMINATION OF KIR HAPLOTYPES ASSOCIATED WITH DISEASE

• US 20110129830A1
• Filed: 09/17/2010
• Published: 06/02/2011
• Est. Priority Date: 09/22/2009
• Status: Active Grant

• Current Assignee: Children's Hospital & Research Center At Oakland, Illumina Incorporated, Roche Molecular Systems (Roche Holding AG)
• Original Assignee: Roche Molecular Systems (Roche Holding AG)
• Inventors: Goodridge, Damian, Trachtenberg, Elizabeth, Bentley, Lloyd Gordon, Erlich, Henry A., Ladner, Martha
• Granted Patent: US 9,005,894 B2
• Time in Patent Office: Days
• US Class Current: 435/6.11
• CPC Class Codes: C12Q 1/6881 for tissue or cell typing, e.g. human leukocyte antigen [HLA] probes
• CPC Class Codes: C12Q 1/6883 for diseases caused by alterations of genetic material
• CPC Class Codes: C12Q 2600/156 Polymorphic or mutational markers
• CPC Class Codes: C12Q 2600/16 Primer sets for multiplex assays
• CPC Class Codes: C12Q 2600/172 Haplotypes
• CPC Class Codes: Y02A 90/10 Information and communication technologies [ICT] supporting adaptation to climate change, e.g. for weather forecasting or climate simulation

Abstract

Disclosed is a method of determining KIR genotypes for one or more individuals in parallel, the method comprising: for each individual, amplifying the polymorphic exon sequences of the KIR genes, pooling the KIR amplicons, performing emulsion PCR followed by pyrosequencing in parallel to determine all the amplicon sequences present in the individual to determine which KIR alleles are present in the individual.

Claims

1. A method of determining KIR genotypes for one or more individuals in parallel, the method comprising:

(a) for each individual, performing an amplification reaction with a forward primer and a reverse primer, each primer comprising an adapter sequence, an individual identification sequence, and a KIR-hybridizing sequence, to amplify the exon sequences of the KIR genes that comprise polymorphic sites to obtain KIR amplicons;

(b) pooling KIR amplicons from more than one individual obtained in step (a);

(c) performing emulsion PCR;

(d) determining the sequence of each KIR amplicon for each individual using pyrosequencing in parallel; and

(e) assigning the KIR alleles to each individual by comparing the sequence of the KIR amplicons determined in step (d) to known KIR sequences to determine which KIR alleles are present in the individual.

2. The method of claim 1, further comprising determining the KIR haplotypes present in each individual.

3. The method of claim 1, wherein step (a) further comprises determining concentration of said KIR amplicons.

4. The method of claim 1, wherein at least one primer has the KIR-hybridizing sequence selected from Table 2.

5. The method of claim 1, wherein at least one primer has an individual identification sequence selected from Table 3.

6. The method of claim 1, further comprising step (f) of determining that the individual is predisposed to preeclampsia when allele KIRDL1 has been found in step (e).

7. The method of claim 1, further comprising step (f) of determining that the individual is predisposed to autoimmune disease when allele KIR2DS1 has been found in step (e).

8. The method of claim 1, further comprising step (f) of determining that the individual is a suitable unrelated hematopoietic stem cell donor when group B KIR alleles have been found in step (e).

9. The method of claim 1, further comprising step (f) of determining the individual'"'"'s HLA genotype.

10. The method of claim 9, further comprising step (g) of determining that the individual is predisposed to clearing an HCV infection when allele KIRDL1 has been found in step (e) and allele HLA-C1 has been found in step (f).

11. The method of claim 9, further comprising step (g) of determining that the individual is predisposed to slow progression of HIV infection to AIDS when allele KIR3DS1 has been found in step (e) and allele HLA-Bw4 has been found in step (f).

12. The method of claim 9, further comprising step (g) of determining that the individual is predisposed to Crohn'"'"'s disease when alleles KIR2DL2 and KIR2DL3 have been found in step (e) and allele HLA-C2 has been found in step (f).

13. A kit for obtaining KIR amplicons to determine KIR genotypes in one or more individuals in parallel, comprising:

a forward primer comprising an adapter region, an individual identification tag and a KIR-hybridizing region;

a reverse primer that comprises an adapter region, an individual identification tag, and a KIR-hybridizing region.

14. The kit of claim 13, wherein at least one primer comprises a KIR-hybridizing region selected from table 2.

15. The kit of claim 13, wherein at least one primer comprises an individual identification tag selected from Table 3.

16. The kit of claim 13, further comprising one or more populations of beads having a primer attached, said primer capable of hybridizing to the adapter regions in said forward and reverse primers.

17. A reaction mixture for obtaining KIR amplicons to determine KIR genotypes in one or more individuals in parallel, comprising a set of primers which includes:

a forward primer comprising an adapter region, an individual identification tag and a KIR-hybridizing region; and

a reverse primer that comprises an adapter region, an individual identification tag, and a KIR-hybridizing region.

18. The reaction mixture of claim 17, further comprising one or more populations of beads, having a primer attached, said primer capable of hybridizing to the adapter regions in said forward and reverse primers.